Abstract: A new recombinant D-arabinose isomerase (D-AI) was used to convert D-galactose to D-tagatose. Subjected to strain screening in the laboratory, Geobacillus pallidus with strong enzyme production capacity was obtained. The D-arabinose isomerase gene (D-AI) was cloned into expression vector pET-28a, and transformed into E. coli BL21 (DE3). Results revealed that the recombinant strain could express a large number of soluble proteins by SDS-PAGE. Optimum pH and temperature of purified enzyme were 7.0 and 60 ℃, and the enzyme revealed good stability within the range of pH 6.0—8.0 and 30—70 ℃. The addition of Mn2+, Zn2+, Fe2+, Ca2+ and Ba2+ could improve the conversion rate of D-tagatose; in contrast, Mg2+ and Cu2+ could result in the different inhibition degrees of enzyme activity. The substrate D-galactose at the concentration of 18 g/L (containing 5 mmol/L Mn2+), was added to pH 7.0 crude enzyme solution from recombinant cells to react of 60 ℃. Under the optimal reaction time, the maximum conversion rate was 41.6% after 12 h reaction. Therefore, biological activity of the recombinant D-AI was successfully expressed in E. coli, and E. coli (BL21) has potential industrial production of D-tagatose.

Key words: Geobacillus pallidus, D-arabinose isomerase, D-tagatose, expression, transformation